Methods and kits for detecting protein kinases

ABSTRACT

Methods and kits for detecting kinase activity A method for measuring protein kinase activity comprising:  
     (a) providing a first solution comprising ATP and a protein kinase to be tested, and a second solution comprising ATP in the absence of said kinase to be tested;  
     (b) adding a substrate capable of being phosphorylated by the protein kinase to be tested to the first and second solutions of step (a);  
     (c) measuring the concentration of ATP and/or ADP, or the rate of change thereof with respect to time, in each of the reaction mixtures formed in step (b) using a bioluminescence reaction; and  
     (d) using the information about the concentration of ATP and/or ADP to determine the activity of the protein kinase to be tested.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to methods for detecting proteinkinase activity and kits for performing such methods.

[0003] 2. Description of Related Art

[0004] Protein kinases play crucial roles in the modulation of a widevariety of cellular events. These enzymes act by transferring phosphateresidues to certain amino acids in intracellular polypeptides, to bringabout the activation of these protein substrates, and set in motion acascade of activation controlling events including the growth,differentiation and division of cells. Protein kinases have beenextensively studied in the field of tumour biology. A lack of controlledactivity of kinases in cells is believed to lead to the formation oftumours. The pharmaceutical industry is constantly in search of drugsthat target these kinases, to help with the treatment of a wide varietyof tumours. There are at least 1200 protein kinases that are involved inthe regulation of cell functions. They occur as both transmembrane andcytosolic enzymes and they phosphorylate serine, threonine and tyrosineamino acid residues. Based on these substrate specificities the kinasesare divided into two groups, the serine/threonine kinases and tyrosinekinases. This has led to the development of a number of techniques thatfocus on the ability of these proteins to take a phosphate group andattach it to a protein/peptide.

[0005] One of the most widely used techniques is a radio-isotope method,that utilises either ³²P or ³³P gamma phosphates. In the presence of anactive kinase, the labelled phosphate is transferred from the ATP to theprotein or peptide substrate. These assays need to be performed in thepresence of ATP labelled to a high specific activity. This results fromkeeping the concentration of unlabelled ATP in the micromolarconcentration range. Also in order to achieve the required sensitivitythe peptide substrate has to be used at high concentrations (5-20 μM).The increased radioactivity on the resulting phosphoproteins can then bedetected using scintillation counters after capture on phosphocellulosepaper.

[0006] Other methods include immunoprecipitation procedures. Duringthese assays the kinase, ATP and substrate reaction is allowed toproceed and is then stopped using a buffer, such as Laemmli buffer. Theprotein is then run out using SDS/PAGE electrophoresis. The gel is thenblotted onto a nitrocellulose membrane and probed for phosphorylatedsubstrate, using an antibody to the phosphorylated amino acid of choice.The presence of the phosphorylated band can be visualised using asecondary antibody conjugated to horseradish peroxidase, followed by theuse of a chemiluminescence detection system, and exposure ontophotographic film. As in the case of many of the methods that have beenproposed as an alternative to the radioisotope assays, however, theabove western blotting technique lacks sensitivity and is quitelaborious.

[0007] The use of luminescent detection, either by bioluminescence orchemiluminescence allows for a highly sensitive detection system. Lehelet al. (1997) Anal. Biochem. 244, 340-346 reported the use of achemiluminescent microtitre plate assay for detection of protein kinaseactivity. This assay is based on the use of biotinylated substratepeptides captured on a streptavidin-coated microplate, together withmonoclonal antibodies. The authors chose protein kinase A (PKA) todevelop the assay, but also found reliable results with chose proteinkinase C (PKC), calcium/calmodulin-dependent protein kinase II, receptorinteracting protein and src activities. These assays were performed inthe presence of 20 μM ATP and the kinase of interest +/−inhibitor, thekinase reaction was allowed to proceed to completion. The plates werethen washed prior to antibody binding and chemiluminescence detectionwith a secondary antibody conjugated to horseradish peroxidase withchemiluminescence determined using a Tropix (RTM) (USA) chemiluminescentsubstrate kit. This assay still relies upon the availability of specificsubstrates, and also antibodies to the phosphoproteins generated.

[0008] Another approach that has been taken, is to adopt microchip-basedtechnology. Cohen et al. (1999) Anal. Biochem. 273, 89-97, reported anassay for PKA based on photolithographic techniques. Performing anon-chip electrophoretic separation of the fluorescently labelled peptidesubstrate and product allowed for determination of the movement of thephosphate group from ATP to the serine residue of the heptapeptide,Kemptide. This technology was developed for the detection of PKAactivity.

[0009] Eu et al (1999) Anal. Biochem. 271, 168-176 describe a method inwhich the measurement of ATP via bioluminescence is related to theamount of substrate (galactose) which is present in a urine sample.

[0010] Sala-Newby & Campbell (1992) FEBS Lett 307, 241-244 describe theuse of a firefly luciferase which was engineered to contain a proteinkinase A recognition site RRFS and to lack the C-terminal peroxisomalsignal of native luciferase. The mutant luciferase was expressed in COScells and used to detect and quantify protein kinase A activation bycyclic AMP in those cells.

[0011] It will be appreciated that the above method is extremelyspecific, being useful only for protein kinase A activation by cyclicAMP. Hence, it suffers from the same problems as the protein kinasedetection systems described above which are based on the specificenzymes and substrates with which they react.

[0012] There are no assays that have the ability to determine kinaseactivity irrespective of the kinase family or the amino acid residuesthat are phosphorylated. This is due to the fact that all the methodscurrently available focus on the specific enzymes and substratesinvolved.

[0013] The present invention seeks to provide methods for measuringprotein kinase activity which are not specific for a single proteinkinase, but rather can be used as a general means of measuring theactivity of a wide range of protein kinases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1: Shows the drop in ATP light output (relative light units;RLUs) associated with the protein kinase JNK-1. The means of threeseparate experiments ±standard error of the mean (SEM) are shown. Theseare the results from the data generated in the presence of 200 mM Hepes;

[0015]FIG. 2: Shows the effect of Hepes and stop solution on the lightoutput (RLUs) obtained in the presence and absence of activated JNK. Theresults are expressed as the means of two separate experiments,performed in triplicate ±standard deviation (SD). As controls 20 μl ofdistilled water was added in place of stop solution.

[0016]FIG. 3: Shows the effect of MAPK-1/ERK-1 on an increase in ADP.The data are presented as the difference in RLUs, pre and post additionof converting reagent. The results represent the means of 6 differentreplicates for each condition ±SD.

[0017]FIG. 4: Shows the effect of increasing concentrations of MAPK-1 onthe drop in light output. The results are expressed as the means ofthree separate experiments performed in triplicate ±SEM.

[0018]FIG. 5: Shows the initial light output and subsequent signal decayobserved with ATP monitoring reagent and 12 μM ATP in differentbuffering conditions. The results shown are from one representativeexperiment.

[0019]FIG. 6: Shows the effect of JNK-2 activity on the signal decaymeasured over the first two minutes, in the presence of ATP monitoringreagent. The results are from one experiment, representative of three.The assays were performed in triplicate wells of a white 96 wellmicrotitre plate.

[0020]FIG. 7: Shows the effect of increasing concentrations of MAPK-1 onthe decay in light signal. This experiment also compared the effect ofthe activated form of the enzyme versus the inactive form. The resultsare expressed as the means of triplicate experiments ±SD.

[0021]FIG. 8: Shows the effect of different kinase buffers on lightoutput (RLUs) with 1 μM ATP. The data are shown as the means of 6replicate wells ±SD. See Table 1 for buffer details.

[0022]FIG. 9: Shows the time course for the reduction in light outputover time as a result of JNK2α2 kinase activity with ATF-2 as substrate.ADP detection was performed to act as a control confirming cleavage ofthe phosphate group from ATP. JNK2α2 was used at 50 mU and ATF-2 at 5.38μg.

[0023]FIG. 10: Shows a JNK2α2 concentration curve of the reduction inATP with time at 30° C. as an indication of kinase activity. ADPconverting reagent was added to confirm the presence of ADP in thereaction mixture.

[0024]FIG. 11: Shows a comparison of the drop in light signal usingJNK2α2 with c-Jun and ATF-2 as substrates.

[0025]FIG. 12: Shows a SAPK3 concentration curve with a concentrationdependent drop in light signal. The SAPK3 concentrations are shown asnanomolar.

[0026]FIG. 13: Shows the concentration dependent effect of SAPK4 onreduced light output (with MBP substrate). The SAPK4 concentrations areshown as nanomolar.

[0027]FIG. 14: Shows the effect of increasing concentrations of ATPdetection of JNK2α2 activity in the presence of ATF-2 as substrate. Theresults are the means of triplicate wells ±SD.

[0028]FIG. 15: Shows the effect of increasing ATP concentrations onSAPK3 activity in the presence of MBP as substrate. The results areshown as the means of triplicate wells ±SD.

[0029]FIG. 16: Shows the reduction in RLUs in the presence of increasingconcentrations of ATP for SAPK3 and MBP. SAPK3 was used at 728 nM withMBP at a final concentration of 100 μg/ml.

[0030]FIG. 17: Shows a comparison of performance of the kinase assaywhen using 20 μl from a larger reaction volume and when the assay isperformed directly in the wells of a 384 well microtitre plate.

[0031]FIG. 18: Shows the activation of MAPK2 by MEK-1, followed by thephosphorylation of MBP by the previously activated MAPK-2.

[0032]FIG. 19: Shows the correlation of a drop in light signal with theimmunostaining of the phosphorylated MBP by western blotting. The lefthand lane on the blot correlates with the no MBP control, and the righthand lane shows the effect of SAPK3 activity on Upstate Biotechnology(UBI) MBP.

[0033]FIG. 20: Shows a comparison between the bioluminescent assay ofkinase activity and the results of the Western blotting.

[0034]FIG. 21: Shows the effect of staurosporine on the bioluminescentdetection system. The results are presented as the means of triplicatewells SD.

[0035]FIG. 22: Shows the effect of two different staurosporineconcentrations on JNK2α2 activity (with ATF-2 as substrate).

[0036]FIG. 23: Shows the effect of increasing concentrations ofgenistein on MAPK-1 activity (with MBP as substrate).

[0037]FIG. 24: Shows the effect of two different concentrations ofPD098059 on raf-1 activity (with inactive MEK-1 as substrate). Theresults are the means of triplicate wells ±SD.

DETAILED DESCRIPTION OF THE INVENTION

[0038] According to the invention there is provided a method formeasuring protein kinase activity, said method comprising:

[0039] (a) providing a first solution comprising ATP and a proteinkinase to be tested, and a second solution comprising ATP in the absenceof said protein kinase to be tested;

[0040] (b) adding a substrate capable of being phosphorylated by theprotein kinase to be tested to the first and second solutions of step(a);

[0041] (c) measuring the concentration of ATP and/or ADP, or the rate ofchange thereof with respect to time, in each of the reaction mixturesformed in step (b) using a bioluminescence reaction; and

[0042] (d) using the information about the concentration of ATP and/orADP to determine the activity of the protein kinase to be tested.

[0043] The kinase is preferably activated prior to step (a) byphosphorylation. Kinases are involved in very complex intracellularsignalling cascades. On binding of an agonist to a cell membranereceptor a number of phosphorylation events are very rapidly set inmotion. In quiescent cells, a number of kinases are in their inactiveform and require phosphorylation in order to allow these enzymes to thenphosphorylate their substrates. By a domino-like effect, activation ofone component of the pathway may provide a large amplification of thesignal. For example, Raf is a serine/threonine kinase thatphosphorylates and activates MEK (MAPK kinase). MEK, in turn, is a dualtyrosine/threonine kinase which will activate MAPK (Erk-1 and Erk-2) byphosphorylation of the tyrosine and threonine residues. The MAPKs, inturn, are then activated such that they can phosphorylate theirsubstrates, i.e. myelin basic protein.

[0044] Commercially available kinases can be obtained in their activeform (already phosphorylated by the suppliers) or in their inactiveform. The latter require phosphorylation by another kinase which wouldbe upstream from them in the signal transduction pathway.

[0045] Since the methods of the invention are not selective forparticular types of kinases (i.e. serine/threonine vs. tyrosine), theycan be used to monitor the step-wise activation of all the kinases in aparticular pathway, for example by measuring the reduction in ATP seenwhen MEK phosphorylates Erk-1, which in turn phosphorylates myelin basicprotein.

[0046] A second aspect the invention provides a method for identifying acompound which modulates the activity of a protein kinase, said methodcomprising:

[0047] (a) providing a first solution comprising ATP, a protein kinaseand a compound to be tested, and a second solution comprising ATP andthe protein kinase in the absence of said compound to be tested;

[0048] (b) adding a substrate capable of being phosphorylated by saidprotein kinase to the first and second solutions of step (a);

[0049] (c) measuring the concentration of ATP and/or ADP, or the rate ofchange thereof with respect to time, in each of the reaction mixturesformed in step (b) using a bioluminescence reaction;

[0050] (d) using the information about the concentration of ATP and/orADP to determine the activity of the protein kinase in the first andsecond solutions;

[0051] (e) comparing the activity of the protein kinase in the firstsolution with the activity of the protein kinase in the second solutionto identify compounds which modulate the activity of a protein kinase,whereby the compound to be tested is identified as a protein kinasemodulator if the activity of the protein kinase in the first solution isdifferent from the activity of the protein kinase in the secondsolution.

[0052] Exemplary kinase/substrate combinations for use in the methods ofthe invention include JNK-1/cjun, JNK-2/cjun, MAP Kinase-1(ERK-1)/myelin basic protein, MAP Kinase-2 (ERK-2)/myelin basic protein,PKA/Kemptide, MEK-1/inactive MAP Kinase-2 (ERK-2), JNK2α2/ATF-2,JNK2α2/cjun, SAPK-3/myelin basic protein, SAPK4/myelin basic protein andraf-1/inactive MEK-1.

[0053] By “modulate” we include the meaning that the activity of theprotein kinase is increased or decreased or prevented/inhibited in thepresence of the test compound. Thus, the methods of the invention may beused to determine whether a compound is an inhibitor or activator of aprotein kinase.

[0054] The compound to be tested is identified as a protein kinaseinhibitor if the activity of the kinase in the first solution is lowerthan the activity of the kinase in the second solution. Preferably, thecompound to be tested is identified as a protein kinase inhibitor if theactivity of the kinase in the first solution is less than 90% of theactivity of the kinase in the second solution. More preferably, thecompound to be tested is identified as a protein kinase inhibitor if theactivity of the kinase in the first solution is less than 80%, 70%, 60%,50%, 40%, 30%, 20% or 10% of the activity of the kinase in the secondsolution. Most preferably, the compound to be tested is identified as aprotein kinase inhibitor if the activity of the kinase in the firstsolution is less than 50% of the activity of the kinase in the secondsolution.

[0055] Likewise, the compound to be tested is identified as a proteinkinase activator if the activity of the kinase in the first solution ishigher than the activity of the kinase in the second solution.Preferably, the compound to be tested is identified as a protein kinaseactivator if the activity of the kinase in the first solution is morethan 10% greater that the activity of the kinase in the second solution.More preferably, the compound to be tested is identified as a proteinkinase inhibitor if the activity of the kinase in the first solution ismore than 20%, 30%, 40%, 50%, 75%, 100% or 200% greater than theactivity of the kinase in the second solution. Most preferably, thecompound to be tested is identified as a protein kinase activator if thekinase in the first solution at least 50% greater than the activity ofthe kinase in the second solution.

[0056] Conveniently, the first and second solutions of step (a) of themethods of the invention are substantially cell-free.

[0057] Steps (a) to (d) of the method according to the second aspect ofthe invention may be repeated one or more times using a different kinaseand its corresponding substrate each time.

[0058] Compounds which increase the activity of a protein kinase mayfind utility in medicine, especially the study of cancers and may beuseful as therapeutic agents. Compounds which decrease orprevent/inhibit the activity of a protein kinase may also find utilityin such applications.

[0059] Preferably, the first and second solutions of step (a) comprise abuffer, conveniently Hepes buffer.

[0060] Advantageously, steps (a) to (c) are carried out consecutively.

[0061] It will be appreciated by persons skilled in the art that,following addition of the substrate in step (b), the reaction may beallowed to proceed for various durations and at different temperaturesprior to step (c). Advantageously, following addition of the substratein step (b), the reaction mixture is allowed to react for 10 minutes at30° C. prior to step (c). Conveniently, following addition of thesubstrate in step (b), the reaction mixture is allowed to react for 10minutes at 30° C. or for 1 hour at room temperature prior to step (c).

[0062] Preferably, step (c) of the methods of the invention comprises:

[0063] (i) adding a bioluminescent reagent comprising luciferin or aderivative thereof and a luciferase to said reaction mixtures, saidluciferin or a derivative thereof emitting light in a bioluminescentreaction with the luciferase in the presence of ATP; and

[0064] (ii) measuring the intensity of light emitted by the resultantbioluminescent reaction, or its change with respect to time, as ameasure of ATP concentration.

[0065] The bioluminescent reagent of step (c) can be any of theluciferin/luciferase general type. The active substrate is D-luciferinor a derivative thereof. U.S. Pat. No. 5,374,534 discloses D-luciferinderivatives which may be used with luciferase in the methods of theinvention. Any other derivative can be used.

[0066] The luciferase enzyme is preferably obtained naturally,especially from fireflies and most especially Photinus pyralis. However,the source of the luciferase is not critical, so long as it reacts withluciferin (or a derivative thereof) and ATP in the bioluminescentreaction. Examples are luciferases from Luciola cruciata, Diptera spp.and Coleoptera spp.

[0067] Synthetic, for example, recombinant luciferase can be used in theinvention. It is described by Devine et al., (1993) Biochemica etBiphysica Acta 1173, 121-132 and in European Patent No 0 301 541 andU.S. Pat. No. 5,583,024.

[0068] Mutant luciferases may also be used in the methods of theinvention (see below).

[0069] In a preferred embodiment, the method comprises a further step(b′), carried out after step (b) and before step (c), of adding areagent to the reaction mixture formed in step (b) which stops thereaction of the kinase with the substrate.

[0070] There are a number of acids that are suitable for use as a stopreagent, for example commonly used laboratory acids such as phosphoricacid. Alternatively, high concentrations of EDTA or EGTA may beemployed. Luciferase is more resistant to the effects of EDTA/EGTA thanother enzymes, and performance in high concentrations of these salts isincreased by the use of mutant luciferase enzymes. In addition, anyother known buffer for stopping enzyme reactions may be used.

[0071] The stopping reagent is preferably phosphoric acid, EDTA or EGTA.

[0072] The use of a stopping reagent is particularly advantageous as itallows one to make up and store large numbers of samples prior totesting. This feature is particularly desirable for high throughputapplications of the methods of the invention (see below).

[0073] The method of this preferred embodiment advantageously comprisesa further step (b″), carried out after step (b′) and before step (c), ofadjusting the pH of the mixture formed in step (b′) to a pH at which theluciferase enzyme is active, normally pH 7.0. Preferably, step (b″)comprises adding Hepes buffer.

[0074] The step of pH adjustment can be avoided by the use of a mutantluciferase which retains the required activity at the pH of the solutionfollowing addition of the stopping reagent. It is also useful to employa luciferase which is active at 30° C., rather than wild type luciferasewhich is not very active above 25° C. An additional benefit of usingthermostable luciferase mutants is that, in addition to their resistanceto elevated temperatures, is that key amino acids within the enzyme canbe mutated so as to confer other favourable properties that enhance theperformance of the enzyme, including resistance to low pH and high saltsolutions. These properties are therefore helpful if a stop solution isto be used.

[0075] Suitable mutant luciferases can be obtained from KikkomanBiochemicals, Japan.

[0076] Other exemplary mutant luciferases suitable for use in themethods of the invention are disclosed in White et al. (1996) Biochem J.319, 343-350, Squirrel et al. (1997) J. Defence Science 2, 292-297, Karp& Oker-Blom (1999) Biomolecular Engineering 16,101-104, Branchini et al(1999) Biochemistry 38, 13223-13230, Branchini et al. (2000)Biochemistry 39, 5433-5440, Tatsumi et al. (1996) Anal. Biochem. 243,176-180, WO 98/46729, WO 96/22376, WO 99/02697, EP 0 449 621 B, U.S.Pat. No. 5,330,906, U.S. Pat. No. 6,074,859, and WO 95/18853.

[0077] Advantageously, step (c) further comprises the following stepscarried out after the light intensity measured in step (ii) has reacheda substantially constant level:

[0078] (iii) adding a reagent that converts ADP to ATP;

[0079] (iv) adding a bioluminescent reagent comprising luciferin or aderivative thereof and a luciferase to said reaction mixture of step(iii); and

[0080] (v) measuring the intensity of light emitted by the resultantbioluminescent reaction

[0081] wherein the difference in the intensity of light in step (v) andthe steady state intensity of light in step (ii) is a measure of ADPconcentration in the reaction mixture of step (ii).

[0082] By “substantially constant” we include the meaning that the lightintensity does not vary significantly over the same time period as istaken to carry out the light intensity measurements. As a non-limitingexample, the term is intended to include the meaning that the rate ofchange of emitted light intensity is less than 5% per minute, andpreferably less than 3% per minute. In any event the person skilled inthe art will be able to appreciate whether the level is sufficientlyconstant to be able to obtain a valid reading of the ATP produced byadding the ADP-converting reagent, not significantly affected by anysmall change in the ATP baseline.

[0083] In an alternative preferred embodiment, steps (b) and (c) arecarried out simultaneously.

[0084] According to a further aspect, the invention provides a compoundidentified using a method of the invention.

[0085] It will be appreciated by persons skilled in the art that themethods of the invention are suitable for high throughput screening,i.e. screening of large numbers of chemically generated andnaturally-derived products for generating leads to pharmaceuticalproducts. In such screening assays, compounds may be put into groups forscreening using microtitre plate technologies.

[0086] Thus, the methods of the invention can be performed in the smallvolumes associated with 384 and 1536 well plates, in addition to the 96well plate format. Under these circumstances, where laboratory robotsare used, the assays would be prepared in a large number of plates. Theassays could then be carried out using the robots to transport theplates into a luminometer with injectors, and the assay performed asdescribed above.

[0087] Another option arises due to the long half-life of the ‘glow’ oflight from the bioluminescence reaction. Once the reaction hasplateaued, the emitted light intensity remains substantially constant.This allows for the bioluminescent reagent to be added to the plates inbatches, so the plates can be read even 3-4 hours after addition of thereagents.

[0088] The invention further provides a kit for use in the method of thesecond aspect of the invention, comprising:

[0089] (a) a bioluminescent reagent comprising luciferin or a derivativethereof and a luciferase, said luciferin or a derivative thereofemitting light in a bioluminescent reaction with the luciferase in thepresence of ATP;

[0090] (b) a kinase;

[0091] (c) a substrate capable of being phosphorylated by said kinase;and

[0092] (d) ATP.

[0093] The kit conveniently further comprises one or more buffers forreconstituting, diluting or dissolving the bioluminescent reagent,kinase, substrate and/or ATP.

[0094] The kit may also further comprise a reagent capable of stoppingthe reaction of said kinase with said substrate, for example phosphoricacid.

[0095] A kit according to the invention may additionally comprise one ormore reagent(s) which converts ADP to ATP, such as one which comprisespyruvate kinase and phosphoenol pyruvate.

[0096] In a preferred embodiment, the kit of the invention comprises twoor more different kinases and their substrates. Thus, there is envisageda kit suitable for screening compounds to be tested against a pluralityof different kinases to determine whether the compound modulates kinaseactivity and the specificity of such inhibition.

[0097] A kit according to the invention may further comprise amulti-well microtitre plate. This term is intended to embrace apparatuswhich comprises a plurality of reaction vessels or wells linked togetherin the form of a plate. Each well has a small volume, usually 250 to 300μl in a 96 well plate, 60 to 70 μl in a 384 well plate and 6-8 μl in a1536 well plate. At present, the most common plates have 96 wells, butplates having 384 and 1536 wells are known and useful according to theinvention. Preferably, the kit comprises a multiwell microtitre platecontains 96 wells or more.

[0098] Advantageously, the reagent or reagents in a kit of the inventionis or are provided in lyophilised form.

[0099] Examples which embody certain aspects of the invention will nowbe described by way of a non-limiting illustrations which refer to thefigures.

EXAMPLES

[0100] We have performed a series of experiments to show the effect ofprotein kinase activity in a cell free system. All the protein kinasesand substrates were supplied by Upstate Biotechnology Inc., (UBI) LakePlacid, USA. Any other reagents used in a number of differentformulations are shown in appendix 1.

Example 1 Determination of the Activity of JNK-1

[0101] The first set of experiments was to determine the activity ofJNK-1 after the activation of this enzyme by two other kinases, MEKK1and MKK4. The assay buffer used to activate the enzymes was made up as a10 times stock (the formulation is shown in appendix 1). The assay wasperformed as follows, with an initial preparation of a pre-mix for JNKactivation.

[0102] The JNK enzyme was used at a stock concentration of 1.234 mg/ml,MEKK1 at 1 mg/ml and MKK4 at 0.28 mg/ml. The activation mixture wasadded to a polypropylene tube (Sarstedt) with 8.7 μl of JNK, 2.5 μl ofMEKK1, 8.4 μl of MKK4, 1 μl of 10 mM ATP (Calbiochem, UK), 1 μl ofdithiothreitol (Sigma, UK), 5 μl of 10× assay buffer and 23.4 μl ofdistilled water. This was incubated overnight at room temperature. Toperform the actual assay, the 50 μl of activated concentrate was dilutedin 9 mls of JNK assay buffer at the working concentration (i.e. diluted1:10). The substrate used was GST-c-jun at a concentration of 8.61mg/ml. Briefly, 12 μl of GST-cjun was diluted in 1545 μl of workingstrength assay dilution buffer. Each assay point was performed intriplicate wells of a white, opaque, 96 well microtitre plate (Dynex).To each well was added 15 μl of the substrate mix, followed by 30 μl ofthe activated JNK enzyme. The reaction was then allowed to proceed atroom temperature for one hour. The reaction was then stopped by theaddition of 20 μl of 2% (v/v) phosphoric acid (Sigma, UK). To one set oftriplicate wells was added 135 μl of Tris-acetate buffer (pH7.75, seeappendix 1 for formulation), to a second set of triplicate wells wasadded 135 μl of 200 mM Hepes buffer (pH 7.75, see appendix 1 forformulation). After this 20 μl of ATP monitoring reagent (see appendix 1for formulation) was added to each well. The plate was then placedimmediately into a Microbeta (RTM) Jet luminometer (Perkin-Elmer LifeSciences), and the light output was determined over a 1 second integral.FIG. 1 shows the drop in light output seen in the presence of thekinase.

[0103] Stop Solution

[0104] These experiments were performed with 2% phosphoric acid to haltthe kinase reaction at a set time point. A particular advantage of theuse of a stop solution, that is, any reagent which can halt the reactionof the kinase and the substrate which it phosphorylates, is that one canmake up and store large numbers of samples prior to testing in themethods of the invention. This feature is a particular benefit forhigh-throughput screening applications. One of the problems with thestop solution is that a reduction in the pH adversely affects theluciferase enzyme, so there must be sufficient buffering capacity whenthe ATP is measured. In this first series of experiments Hepes proved tobe a much better buffer for counteracting the effects of the phosphoricacid. Experiments were performed with increasing concentrations ofHepes, and showed that the 200 mM buffer brought the pH in the well backto pH 7.0 allowing the luciferase-luciferin reaction to proceed withoutreactivating the kinase. The Tris acetate buffer was less efficient, andwhile there were still differences in the detected RLUs in the presenceof the kinase enzyme, however, they were not as marked as with theHepes. The results with Hepes showed lower RLUs than without, but thisapproach did allow for the kinase activity to be controlled prior tobeing detected using the luciferase-luciferin reaction. As a result ofthis, most of the following experiments were performed with the 200 mMHepes, as a dilution buffer, unless otherwise stated. FIG. 2 shows thedata from two separate experiments and demonstrates the effects of boththe phosphoric acid stop solution and the Hepes buffer.

[0105] Skilled persons will appreciate that one may avoid the use of abuffer after addition of the stop solution by using mutant luciferaseswhich are pH and salt stable. It is also desirable to use mutantluciferase which are thermostable at 30° C. rather than wild typeluciferase, which is not very active above 25° C. Suitable mutants areavailable from a variety of sources. For example, pH, salt stable andthermostable mutants can be acquired from Kikkoman Biochemicals, Japan(see above).

Example 2 Determination of the Activity of MAPK-1/ERK-1

[0106] To confirm that monitoring of kinase activity by the above methodof the invention would proceed with other enzymes that cleave thephosphate from ATP, we looked at a number of other kinases which areimportant in signal transduction and used as targets in drug discovery.

[0107] MAP Kinase-1/ERK-1 activity was investigated in the presence ofmyelin basic protein as the phospho-acceptor. This enzyme was suppliedin the active form from UBI at a stock concentration of 25 μg in 250 μl.Briefly, 10 μl of assay dilution buffer (UBI, see appendix 1 forformulation) was added to triplicate wells of a white-walled 96 wellmicrotitre plate (Dynex).

[0108] To this was added 10 μl of MAPK1 and 10 μl of myelin basicprotein (UBI, 2 mg/nl), plus 10 μl of ATP cocktail (UBI, for formulationsee appendix 1). The plate was sealed and the reaction was allowed toproceed for 10 minutes at 30° C. After this time 110 μl of either assaydilution buffer or Hepes buffer were added to the wells followed by 20μl of ATP monitoring reagent reconstituted in 200 mM Hepes buffer. Theresults showed a reduction in light output with this enzyme in thepresence of its substrate. With the UBI buffer there was a drop in RLUsfrom 77367 to 35578, with the Hepes buffer there was a reduction from91256 to 73424.

[0109] Detection Based on Increase in ADP

[0110] With this experiment we also determined whether it would bepossible to detect any resultant increase in ADP by the conversion ofADP to ATP, through the addition of 20 μl of an ADP converting reagentcontaining pyruvate kinase (for formulation see appendix 1). Todetermine the amount of ADP, a reading was taken after the initial ATPlight signal had been allowed to decay for 10 minutes. The convertingreagent was then added and a further reading taken after 5 minutes. Allof these readings were integrals taken after 1 second using theMicrobeta (RTM) Jet (Perkin-Elmer Life Sciences). The amount of ADPpresent correlated with the difference in light output between the finalreading and the reading taken prior to the addition of convertingreagent. The data showed that it was possible to determine an increasein ADP, as shown in FIG. 3.

Example 3 Determination of the Activity of MAPK-2/ERK-2

[0111] The above protocol was also used for determining the effect ofMAPK-2/ERK-2 in the presence of the same substrate, myelin basicprotein. The MAPK-2 was supplied by UBI at a concentration of 2.5 μg ofenzyme in 25 μl of buffer (for formulation see appendix 1), with aspecific activity of 662.5 U/mg, where 1 U=1 nmole of phosphateincorporated into myelin basic protein. This enzyme was compared withthe inactive form, also supplied by UBI at a concentration of 12.5 μg in50 μl. Both enzymes were diluted in UBI assay dilution buffer to allowfor the addition of 25 ng of protein in 10 μl to each well. The myelinbasic protein was added as described in the above example, and 200 mMHepes was used as the buffer added to the wells prior to the addition ofthe ATP monitoring reagent. The assay was performed in triplicate wellsand showed RLUs of 10075±339 for the inactive enzyme, which was verysimilar to the light output from the no enzyme controls (11440±1372).The RLUs for the active enzyme showed a drop in ATP, after the 10minutes incubation, to 7008±430. It was also possible to detect anincrease in RLUs after addition of ADP converting reagent in the activesample of 5272, compared with 441 with the inactive enzyme.

Example 4 Effect of Kinase Concentration on Reduction in ATP

[0112] To determine whether there was a concentration dependent effectof kinase activity on reduction in ATP, MAPK-1 was used atconcentrations ranging from 1.56 ng/10 μl to 100 ng/10 μl, with serialdoubling dilutions from the highest concentrations. The concentration ofmyelin basic protein used was the same as in the previous experiments.The assays were performed as in the previous two experiments, i.e. usingUBI reagents, but with the addition of 110 μl of 200 mM Hepes bufferprior to determination of ATP readings. The results showed aconcentration dependent reduction in ATP levels detected with increasingconcentrations of the MAPK-1. There was no effect of the enzyme when1.56 ng was added to each well, however, there was a significant effectat concentrations of 3.13 ng and above (see FIG. 4).

Example 5 Effect of ATP Concentration on Light Decay: Influence ofBuffer Type

[0113] The luciferase enzyme itself is an ATPase that converts ATP toAMP and inorganic phosphate. After the initial increase in light outputas a result of the luciferin-luciferase reaction, the light signalbegins to decay over time. We examined the light decay with increasingconcentrations of ATP up to the 200 μM used in the above experiments.The ATP standards (Sigma, UK) were diluted at serial doubling dilutionsfrom 200 μM down to 3.125 μM per 10 μl added to each well. The standardswere diluted in three different buffers, assay dilution buffer from UBI,Tris acetate buffer (pH 7.75) and 200 mM Hepes pH 7.7. 10 μl of thestandards, plus a no ATP buffer control, were added in to triplicatewells of the opaque-white 96 well microtitre plates. The experimentswere run with duplicate plates, one where 1401 μl of the Tris-acetatebuffer was added to all wells, and the other where the same volume of200 mM Hepes buffer. Immediately after the addition of these reagents 20μl of ATP monitoring reagent was added to all wells. The plate was thenplaced in a Berthold (RTM) Detection Systems MPL2 luminometer, and theprogram was set to take 1 second integral readings for each well every 2minutes. The FIG. 5 graph shows the initial light output (at time 0) andthe decay in the light signal observed with the different bufferconditions and the 200 μM stock ATP (final concentration in the well of12 μM).

Example 6 Effect of Luciferin-Luciferase (ADR) Reagent on Kinase Assay:Detection of Kinase Activity as a Drop in Light Signal

[0114] Experiments were also carried out to investigate the effect ofperforming the kinase assay in the presence of luciferin-luciferasereagent (ADR), and its effect upon the rate of signal decay. The enzymeused was JNK-2 (Upstate Biotechnology Inc, USA) at 1 μg per 10 μl addedto each well, the c-jun (1-169)-GST substrate (Upstate BiotechnologyInc, USA) was also added at the same concentration. Into each well wasadded 10 μl of 200 μM ATP standard and 10 μl of Hepes buffer (200 mM).Into control wells the 10 μl of enzyme was replaced by 10 μl of Hepesbuffer, once all the active reagents were in the wells and an additional120 μl Hepes dilution buffer had been added, then 20 μl of ADR was addedand the signal decay monitored every 20 seconds over a 2 minute period,using a Berthold (RTM) Detection Systems MPL-2 luminometer. A 1 secondintegral reading was taken at each time point. The data showed anincrease in the rate of signal decay in the presence of the JNK-2enzyme, as shown in FIG. 6. This shows that kinase activity can also bedetected as an accelerated drop in light signal, in the absence of astop solution.

Example 7 Comparison of Active and Inactive Forms of a Kinase

[0115] We also compared activated and inactive forms of MAPK-1 andMAPK-2, and showed reduced kinase activity with the inactive forms ofthe enzymes, although in some cases there did appear to be a certainamount of autophosphorylation, this was not significantly different fromthe no enzyme controls. A concentration curve for inactive MAPK-1 versusthe activated form, showed clearly how the kinase activity of the enzymereduced the amount of ATP and hence increased the signal decay. Theexperiment was performed as detailed in Example 3, however in this casethe kinetics of the reaction were investigated over the first 6 minutesof the reaction. From FIG. 7, it can be seen that at 100 ng of MAPK-1per 10 μl (588 ng/ml final concentration) gave a significant increase inthe % signal decay by 6 minutes. The assay was performed in triplicatein 3 different experiments. As soon as the ADR had been added the platewas read for 1-second integrals every minute for 6 minutes, using aLabsystems Luminoskan (RTM) luminometer.

[0116] A similar effect was also seen with the phosphorylation of MAPK-2by MEK-1. In this series of experiments the MEK-1 was used at finalconcentrations in the well of 75 μg/ml through to 375 μg/ml by theaddition of 1-5 μL of stock enzyme at 5 U/50 μl. The activity quotedfrom Upstate Biotechnology Inc was 7850 U/mg where 1 unit will maximallyactivate 1 unit of inactive MAPK-2. The inactive MAPK-2 was added toeach well in 10 μl volumes to give a final concentration of 5.88 mg/ml.Again, in the presence of 200 μM ATP and Hepes buffer, there was aconcentration dependent increase in the signal decay from 14% in thecontrol to 36% for the highest concentration used.

Example 8 Effect of Different Kinase Buffers

[0117] In the literature there are a number of different reactionbuffers that are used to perform kinase assays, we therefore decided totest the ATP detection reagent with these buffers, to ensure that theassay would perform irrespective of the constituents of the buffer. Thereason these buffers are used is to supply optimal conditions for thekinase reaction in the presence of the ATP and protein/peptidesubstrates. FIG. 8 shows the effect of 13 different buffers commonlyused in protein kinase assays. The buffer constituents are shown inTable 1. TABLE 1 Used for Buffer i.d. Buffer contents Kinases 0 100 mMTris Hcl pH 7.4 containing 20 mM DTT, 20 mM MnCl₂, 10% Glycerol SBE Mix(Astrazeneca) and 0.004% Brij-35 1 8 mM MOPs pH7.0 containing 0.2 mMEDTA GSK3β, S6k1, MAPKAP- K1b/RSK2, PKA, CHK1 CHK2, MSK1 and SGK 2 50 mMTris-Hcl pH7.75 containing 0.05% 2-Mercaptoethanol PKBα 3 25 mM Tris-HclpH 7.5 containing 0.1 mM EGTA ERK-2 SAPk2α/p38, SAPK2b/p38/p32, SAPk3and SAPk4 4 50 mM Sodium β glycerophosphate pH7.5 containing 0.1 mM EGTAMAPKAP-K2 and PRAK 5 50 mM Tris-Hcl pH 7.5 containing 0.1 mM EGTA and0.1% 2-Mercaptoethanol. JNK-1, ROCKII and PRK-2 6 50 mM Hepes pH7.4containing 1 mM DTT, 0.02% Brij-35, and 0.2 mM AMP. AMPK Respiratory 720 mM Hepes pH 7.4 containing 0.03% Triton X-100, 0.1 mM Cacl₂, 0.1mg.ml PKCα phosphatidylserine and 10 μg.ml 1,2-dioleoyl-sn-glycerol. 820 mM Hepes pH7.4 containing 0.03% Triton X-100, 0.1 mM EGTA, 0.1 mg.mlPKCδ phosphatidylserine, 10 μg.ml 1,2-dioleoyl-sn-glycerol 9 20 mM HepespH7.6 containing 150 mM NaCl, 0.1 mM EDTA, 5 mM DTT and CK2 0.1% TritonX-100 10 50 mM Tris-Hcl pH7.5 containing sodium β-glycerophosphate, and0.04 mM PHK Cacl₂ 11 25 mM Tris-Hcl pH7.5 containing 0.1 mM EGTA, 0.1%2-Mercaptoethanol and ERK2 0.01% Brij-35 12 50 mM Tris-Hcl pH7.5containing 0.1% 2-Mercaptoethanol PDK-1 13 50 mM Tris-Hcl pH7.5containing 0.1 mM CaCl₂, 10 units per ml calmodulin MLCK and 0.1% 2Mercaptoethanol. 14 50 mM Tris-Hcl pH7.5 containing 0.1 mM EGTA, 0.1 mMNa₄Vo₃ LCK 14

[0118] The ATP was diluted in each of the buffers and 100 μL was addedto each well of a 96 well microtitre plate, 20 μl of ATP detectionreagent was added to the wells, and the light output was detected aftera 1 second integral reading using a Berthold (RTM) Detection SystemsOrion Luminometer.

[0119] The results in FIG. 8 show that buffers 0 and 9 compromise thelight output, however the sensitivity of the assay was unaffected. Theseassays are normally performed with ATP concentrations from tens tohundreds of micromolar, i.e. greater than the 1 μM concentration used inthis example, where a significant light output was still detectable withthe buffers that reduced the signal.

Example 9 Use of Tris Acetate Buffer in Bioluminescence Assay

[0120] We have compared the performance of the bioluminescent assay inTris buffer as well as Hepes. We have shown that it is possible toreconstitute the ATP Detection Reagent in Tris Acetate Buffer at pH7.75. However, when using an acid stop solution, for example phosphoricacid, it is preferable to use the Hepes buffer reconstitution systemdescribed above.

[0121] The assays were performed using a number of different kinases andsubstrates. The experiments were carried out using methods similar tothat described for Hepes buffer. The assays were performed in 100 μlvolumes in wells of a 96 well microtitre plate, the appropriateconcentrations of each enzyme and substrate were added to the wells inthe most suitable reaction buffer for that enzyme. The reaction was theninitiated by the addition of ATP at the appropriate concentration, thereaction was allowed to proceed at 30° C. for 10 minutes prior toaddition of 20 μl of ATP detection reagent (reconstituted in Trisacetate buffer), light output was detected over a 1 second integral. Inthe following examples, the ATP detection reagent was reconstituted inTris-acetate buffer pH 7.75, rather than a Hepes.

[0122] JNK2α2 with ATF-2 and c-Jun as Substrates

[0123] This enzyme was tested with both the ATF-2 and c-Jun substratepeptides from Upstate Discovery. Assay Buffer: 100 mM Tris-HCl pH 7.4 20 mM Dithiothreitol  20 mM MgCl₂   10% glycerol 0.004% Brij 35

[0124] Time course experiments were performed in 100 μl (or 200 μl)volumes in clear plastic test tubes. The reaction was carried out ineither a 30° C. waterbath or in an incubator.

[0125] Upstate Discovery recommend use of the ATF-2 substrate at 5 μgper assay point and the JNK2α2 at 20 mU per assay point.

[0126] Stock ATF-2 (LB018LFp107) at 4.3 mg/ml in 10 μl aliquots (43 μgin 10 μl). The substrate was diluted 1:8 from stock and then 10 μl addedto the tube to give a final amount in the reaction mixture of 5.38 μg.

[0127] Stock JNK2α2 (LB021SDp81) is at 77 U/mg (100 U/ml) and 1.3 mg/mland aliquoted into 10 μl aliquots (1 U). Concentration curves wereperformed with the addition of 5 μl of neat enzyme stock (50 mU), 5 μlof 1:2 (25 mU), 1:4 (12.5 mU) and 1:8 (6.25 mU), per 100 μl of finalreaction volume.

[0128] Stock ATP at 1M in 10 μl aliquots. The ATP was diluted 1:40 withTris-HCl buffer (addition of 390 μl) and then a further 1:100 to give aworking solution of 250 μM. Then 10 μL was added to each 10011 of finalreaction volume, this gave a final concentration in the tubes of 25 μM.The tubes therefore contained: 10 μl substrate  5 μl enzyme 10 μl ATP 75μl assay buffer

[0129] To determine kinase activity 20 μl samples were removed from thetubes at 5 minute intervals and added to the wells of a 96 well whiteopaque microtitre plate. Then 20 μl of ADR reconstituted in Tris-Acbuffer was added to the wells, and the plate read in the luminometerover a 1 second integral.

[0130] To determine reproducibility a larger volume was prepared andtriplicate samples taken over reduced time points.

[0131]FIG. 9 shows the drop in light output with time in the presence ofenzyme and substrate compared with the substrate only control.

[0132]FIG. 9 also shows the effect of adding ADP converting reagent (20μl), this converted the ADP formed as a result of kinase activity backto ATP, for detection with the ATP detection reagent. The drop in lightsignal after pipetting of the ADP converting reagent was due to a dropin pH in the well after addition. FIG. 9 clearly shows a marked drop inlight output in the presence of enzyme and substrate compared with theno enzyme control.

[0133] As described in the above methods, the assay was repeated withdecreasing concentrations of JNK2α2, against the same ATP and peptideconcentrations. FIG. 10 shows the data obtained with 2.55 μM (50 mU)down to 320 nM JNK2α2.

[0134] In addition to ATF-2 we also studied another JNK substrate,namely the peptide c-Jun. For determining JNK2α2 activity with the c-Junpeptide the experiments were performed using the same protocol as above,but with the addition of 10 μl of c-Jun rather than the ATF-2.

[0135] Stock c-Jun (LB023JTp72) at 4.83 mg/ml in 80511 in 10 μl=48.3 μg,to use approximately the same amount of peptide as ATF-2 again dilute1:9 and then add 10 μl per tube.

[0136]FIG. 11 compares the drop in ATP as a result of kinase activitywhen the two different substrates were compared. The data confirmedinformation received from Upstate Discovery that the ATF-2 was a moreefficient substrate than the c-Jun, for JNK2α2 activity.

[0137] SAPK-3 and Myelin Basic Protein

[0138] SAPK3 is a member of the mitogen activated protein kinase (MAPK)family, which can be activated by a variety of extracellular agonists.These stress activated protein kinases can utilise myelin basic protein(MBP) as the phospho-acceptor in kinase reactions. We have shown thatthis kinase activity can be determined with ADP detection reagentreconstituted in Tris acetate buffer (pH 7.75) in addition to Hepesbuffer. The assays were performed as follows.

[0139] This assay was performed using the same buffer as forMAPK-2/ERK-2, with a 30° C. incubation. ATP was used at the sameconcentration as for JNK2α2. Assay Buffer:  25 mM Tris-HCl pH 7.5  10 mMMg acetate 0.1 mM EGTA

[0140] As with the previous enzyme the assay was set up in tubes inorder to perform a time course. After the kinase reaction was completed20 μl of reaction mixture was placed in the wells of a 96 well opaquewhite microplate, followed by addition of 20 μl of ATP detectionreagent. The light output was again determined over a 1 second integral.

[0141] Tubes contained: 10 μl enzyme, 10 μl substrate, 10 μl ATP and 70μl assay buffer.

[0142] SAPK3 stock solution (LB012SDp174; 220 μl) was provided at aconcentration of 1.55 mg/ml and 87.3 U/mg. The enzyme was aliquoted into10 μl aliquots (15.5 μg). The enzyme was then diluted 1:3 (=5.17 μg/10μl).

[0143] For enzyme concentration curves the enzyme was diluted a further1:5 (1.03 μg) and 1:10 (0.52 μg). The assay was performed by theaddition of 10 μl per 100 μl final reaction volume. The final enzymeconcentrations were 0, 0.517, 1.03 and 5.17 μg/ml (corresponding tonanomolar concentrations of 0, 72.8, 145.6 and 728.0 nM, respectively;see legend to FIG. 12).

[0144] MBP from Calbiochem (0.5 mg/ml) used 10 μl of the neat stock per100 μl final reaction volume (2.72 μM final concentration).

[0145] Results of the SAPK3/MBP experiment are shown in FIG. 12, inwhich a concentration dependent drop in ATP (as measured by lightoutput) is evident.

[0146] SAPK4 and Myelin Basic Protein

[0147] The above experiment was also repeated with SAPK4 and MBP. Theseassays were performed using the same assay buffer as for MAPK2/ERK-2 andSAPK3.

[0148] Stock SAPK4 (LB012SDp176) at 1.64 mg/ml and 144.1 U/mg in 1271μl. This was aliquoted into 5 μl samples (8.2 μg/aliquot). To generate aworking stock the enzyme was diluted 1:4 (2.05 μg/5 μl), then further1:5 (410 ng/5 μl) and 1:10 (205 ng/5 μl). Then 5 μl was added to eachtube, this gave final concentrations in the tubes, for a 100 μl reactionvolume of 0, 0.103, 0.205 and 1.25 μg/ml (corresponding nanomolarconcentrations are shown in the FIG. 13).

[0149] Stock MBP, Life Technologies at 2.5 mg/ml. 10 μl was added toeach 100 μl final reaction volume (250 μg/ml or 13.6 μM).

[0150] The higher concentration of MBP showed a rapid decrease in thelight signal at time 0, this was in fact approximately 2 minutes afterthe addition of the reagents, as it took this long to remove samplesfrom the tubes, plate them out and then add the ATP detection reagent(see FIG. 13).

[0151] The work performed with the SAP kinases also showed that it waspossible to use MBP from a number of different suppliers (although theperformance of the assay was found to depend upon the quality of theprotein provided).

[0152] These data confirm that Tris-acetate buffer could be used for ATPdetection reagent reconstitution as well as the Hepes buffer describedpreviously.

Example 10 Effect of ATP Concentration

[0153] All of the above assays were performed with ATP at a finalconcentration of 25 μM. We went on to investigate whether thebioluminescent system could detect kinase activity with higher and lowerATP concentrations. The assays were performed with the same buffers asdescribed above, and with the same volumes. However, in the followingexamples the experiments were performed in the wells of white-walled 96well microtitre plates, rather than in tubes. After 30 minutes at 30°C., the plates were removed from the incubator and 2011 of ATP detectionreagent was added to each well and the light out put read over a 1second integral.

[0154] JNK2α2

[0155]FIG. 14 shows the data obtained with ATP at 3 different finalconcentrations 1, 10 and 100 μM (results are shown as means oftriplicate wells ±SD). The substrate used was ATF-2 at a finalconcentration of 2.1 μM, with enzyme at 1.25 μM. The assay buffer wasthe same as that described for JNK2α2 above.

[0156] SAPK3

[0157] The ATP concentration curve experiments were repeated with SAPK3and MBP as substrate. FIG. 15 shows the effect of different. ATPconcentrations on the change in light output (results are shown as themeans of triplicate wells +SD). The SAPK3 was used at a concentration of728 nM, with myelin basic protein (MBP) at a final concentration of 2.72μM in the wells. At 100 μM there was an effect of the enzyme in thepresence of the substrate where the light signal dropped by 693,234RLUs, from 5,267,900±133,688 to 4,574,666±283,204. Th is was asignificant decrease in RLUs and indicated that the amount of enzyme andsubstrate was limiting at this high concentration of ATP.

[0158] The differences in RLUs correlate directly with ATPconcentrations this drop in light out put relates to the amount of ATPdephosphorylated by the SAPK3. This is further demonstrated in FIG. 16,where the differences in RLUs are shown, which indicates that the sameamount of ATP was consumed at 100 μM ATP as 10 μM.

[0159] Although it is not clear from FIG. 16, there was also asignificant difference in RLUs with the lowest concentration of ATPused, where the RLUs dropped from 818±18 to 300±17 (means±SD).

Example 11 Assay Performance in 384 Well Microtitre Plates

[0160] The time course experiments described above confirmed that it waspossible to perform the assay in tubes in large volumes, and then ateach time point sample 20 μl for addition to a white opaque microtitreplate (96 wells), with light output being measured after the addition of20 μl of ATP detection reagent. We have also shown that it is possibleto perform the assay in 100 μl volumes in the wells of a 96 well plate.

[0161]FIG. 17 shows a comparison of performance of the assay when using20 μl from a larger reaction volume and when the assay is performeddirectly in the wells of a 384 well microtitre plate. In 384 wells, thekinase reaction is performed in 20 μl volumes with the addition of 20 μlof ATP detection reagent. The data show that there was no difference inthe performance of the assay when carried out in tubes (100 μl) or 384well plates (20 μl).

Example 12 Study of Kinase Cascades

[0162] The activation of kinases is most often the result ofphosphorylation by other kinases upstream in the signal transductionpathways. An example of this is the activation of MAPK-2 by MEK-1,followed by MAPK-2's ability to phosphorylate MBP. We used this systemto confirm that MAPK-2 had been phosphorylated and activated by MEK-1.If the protein had been activated, there would be a reduction in ATPwhen MAPK-2 was subsequently exposed to MBP in the presence of ATP. Theassay was performed in tubes in 100 μl volumes, the initial activationof inactive MAPK2 was performed at 30° C. with 10 μM ATP. The assaybuffer comprised 25 mM Tris acetate pH 7.75 with 0.1 mM EGTA and 10 mMmagnesium acetate. MEK-1 was used at a final concentration of 114 nMwith inactive MAPK-2 at a final concentration of 516 nM.

[0163] To determine kinase activity, 20 μl was then added to duplicatewells of a 96 well plate, and 20 μl of ATP detection reagent added andthe light signal determined over a 1 second integral.

[0164] A second plate had been set up containing triplicate wells with10 μl of MBP (Calbiochem) at 0.25 mg/ml, 10 μl of 10 μM ATP and 60 μl ofassay buffer, to this 20 μl of the MEK-1/MAPK2 reaction mixture from thetubes was added, and the reaction allowed to proceed in an incubator at30° C. for 30 minutes. After this time 20 μl of ATP detection reagentwas added to the wells and the light output was determined.

[0165] The results are shown in the following FIG. 18 wherein anincrease in the RLUs in the substrate only controls is observed, whichrelates to addition of ATP from the original MEK-1/MAPK-2 reactionmixture. The data clearly show that the MAPK-2 had been phosphorylatedand was therefore able to exhibit kinase activity itself in the presenceof MBP. An additional control was run with the inactive MAPK-2 and MBPwhich showed no drop in light signal.

Example 13 Western Blotting Studies of Substrate Phosphorylation

[0166] In addition to showing that we could induce functional activityof kinases, and detect this using the bioluminescent assay, we confirmedthat the drop in light output was associated with phosphorylation ofamino acids on peptide/protein substrates by Western Blotting.

[0167] Methods

[0168] Sample Preparation: After completion of the kinase reaction in100 μl volumes, 20 μl of reaction mixture was added to 20 μl of 2×Laemmli sample buffer (Amersham, Bucks, UK) and heated at 100° C. for 4minutes, before being placed immediately on ice until required.

[0169] A molecular weight HRP protein marker (New England BioLabs) wasprepared by adding 10 μl of marker to 10 μl of Western blotting samplebuffer.

[0170] Gel Electrophoresis (SDS PAGE) and Transfer Blot: 20 μl of eachof the prepared samples (equivalent to 10 μl of kinase reaction mixture)was added into the lane wells of a 12% SDS Ready Gel (BioRad, Herts, UK)and run with a standard Tris-Glycine running buffer for 45 minutes at180 V.

[0171] The gel was equilibrated in standard Tris-Glycine-Methanoltransfer buffer for 5 minutes at room temperature, before transfer tonitrocellulose membrane using the BioRad mini blot apparatus at 100 Vfor 60 minutes.

[0172] Membrane Probing: The membrane was blocked with PierceSuperblock® (IL, USA) blocking buffer for 1 hour at room temperature.Primary antibodies used were supplied by Promega (Wisconsin, USA) forMAPK, p38 and JNK, and by UBI for anti-phosphorylated MBP. Antibodydilutions were made in Superblock® diluted 10 fold in distilled water.The primary antibody was used at 1:10000, and where appropriatesecondary antibodies at 1:20000. The HRP conjugated detection reagentwas used at 1:10000 for detection of the HRP protein marker.

[0173] Chemiluminescent Detection: Probed membranes were incubated with10 mls of SuperSignal® West Pico (Peirce, Ill. USA) for 2 minutes withslight agitation. The probed blots were then exposed to Hyperfilm(Amersham, Bucks, UK) for 20 seconds. Phosphorylated targets werecompared against the molecular weight markers for identification.

[0174] Results

[0175]FIG. 19 shows the effect of SAPK3 activity on MBP after 30 minutesat 30° C. In this experiment, the assays were run in tubes as describedpreviously. For determination of ATP levels, a 201 aliquot of sample wasremoved into duplicate wells of a 96 well luminescent compatible plate,and the remainder used for Western blotting analysis. The blot wasprobed using an antibody to phosphorylated MBP (Upstate Biotechnology).

[0176] The above experiment was repeated using SAPK4 and a number ofdifferent supplies of MBP. The results are shown in FIG. 20, andhighlight that some proteins, even when used at the same concentrationsgave different results with both the bioluminescent assay and Westernblotting. The blots were again probed with the same anti-phosphorylatedMBP. As the results show, one of the batches of MBP used (CB) had noeffect in either detection assay. Two different samples of MBP were usedfrom Upstate Biotechnology, one was the dephosphorylated form (DePhos)and the other standard MBP (UBI). The bioluminescent data suggested thatfor the same amount of protein used (2.72 μM), the dephosphorylated formwas more efficient in the SAPK4 assay. This could not be determined fromthe immunoblotting, confirming that bioluminescence is a more sensitiveand quantitative assay for kinase activity.

[0177] Summary

[0178] The studies described in Examples 1 to 13 above demonstrate theversatility of the methods and kits of the present invention.

[0179] In particular, the methods of the invention may be used to studya number of different protein kinase/substrate combinations. The proteinkinase enzymes used in the above examples included:

[0180] (i) JNK-land JNK-2 in the presence of c-jun peptide as substrate;

[0181] (ii) MAPK-1/ERK-1 and MAPK-2/ERK-2 with myelin basic protein assubstrate;

[0182] (iii) MEK-1 with inactive MAPK-2 as substrate;

[0183] (iv) JNK2α2 with ATF-2 and c-jun as substrates;

[0184] (v) SAPK-3 with myelin basic protein as substrate; and

[0185] (vi) SAPK-4 with myelin basic protein as substrate;

[0186] In addition, comparisons were made between the activated andinactive forms of both MAPK-1/ERK-1 and MAPK-2/ERK-2 in the presence ofmyelin basic protein.

[0187] The results from these experiments show that it was possible todetect kinase activity in the presence of ATP and the appropriate enzymesubstrate, through a decrease in detectable ATP, an increase in measuredADP, and also accelerated signal decay in the presence of luciferase.

[0188] Several advantages of the assays of the invention are summarisedin the following points.

[0189] The assay can be applied to any protein kinase that cleaves aphosphate from ATP.

[0190] The protein kinase activity could be allowed to go to completion,prior to detection of ATP and/or ADP.

[0191] The assay could be performed in the presence of the ATPmonitoring reagent, with protein kinase activity being determined as adrop in light output, together with an increase in signal decay.

[0192] Changes in adenylate nucleotides showed a concentration dependenteffect with variations in enzyme, substrate and ATP concentrations.

[0193] The drop in light signal correlates with protein phosphorylation.

[0194] The methods can be used to detect and study protein kinaseinhibitors.

[0195] The methods can be used to study protein kinase cascade systems.

[0196] The assays could be performed at room temperature or 30° C.

[0197] Changes in adenylate nucleotides could be detected in thepresence or absence of a stop solution, permitting mass screening ofsamples. By using an appropriate buffer (e.g. Hepes), it was alsopossible to stop the protein kinase reaction with 2% phosphoric acid anddetect the reduced amount of ATP as a result of protein kinase activity.

[0198] The methods can be used with a number of different protein kinasebuffers

[0199] The ATP detection reagent can be used in either Tris acetate orHepes buffers.

[0200] The assay can be supplied as a kit. Different kits could besupplied for measurement of drop in ATP, with or without the use of astop reagent. The kits could also contain the ADP converting reagent (asoutlined in appendix 1), for detecting an increase in ADP as a result ofprotein kinase activity.

[0201] Applications of the Methods of the Invention

[0202] The methods and assays of the present invention, as described inthe above examples can be used as a measure of kinase activity in cellfree systems. This is of particular importance in the pharmaceuticalindustry since it enables the methods and assays to be used in highthroughput screening laboratories, e.g. for identification of drugs thatcan act as kinase modulators, especially inhibitors. For thisapplication, the assays can be carried out exactly as described in theabove examples.

[0203] The methods and assays of the invention may also be used todetermine kinase activity in cellular extracts, and to determine theeffect of modulators of cellular kinase activity. To perform theseexperiments, it is possible to substitute the known kinase in the aboveexamples for the cell extract/supernatant. The substrate is added asdescribed, and any changes in kinase activity in cells treated withinhibitors/activators can be detected.

[0204] There is an increasing number of protein kinases that are beingtargeted for the development of new tumour therapeutics. As this listincreases it will become impractical and extremely expensive to usespecific tests for each kinase. The assays and kits of the presentinvention allow for the detection of a wide spectrum of kinases andprovide a common end point detection system for all kinases. This allowsfor greater ease of use, particularly in high throughput screeninglaboratories, where robots can be set up with all the detection reagentsand the various kinases and inhibitors, of interest, and with opaquewhite (or black) luminometer microtitre plates.

[0205] Moreover, the use of a stop solution allows for a large number ofplates to be batched up (i.e. stored) prior to analysis of ATP and/orADP levels.

Example 14 Study of Kinase Inhibitors

[0206] Staurosporine

[0207] We initially chose to look at a broad spectrum kinase inhibitorstaurosporine (Calbiochem). Firstly, we tested the inhibitor (in DMSO)on the ATP detection reagent to ensure that the inhibitor would notaffect the luciferase-luciferin reaction.

[0208]FIG. 21 shows the effect of staurosporine on the bioluminescentdetection system. The results are presented as the means of triplicatewells ±SD.

[0209] Data show that, even at the highest concentration of theinhibitor, there was no significant effect upon light output. Theexperiment was performed using 10 μM ATP in 100 μl volumes in a 96 wellplate with the addition of 20 μl of ATP detection reagent.

[0210] The effect of staurosporine was tested on the JNK2α2 enzyme withATF-2 as substrate, and as the following figure shows the expectedinhibitory activity could be detected using the bioluminescent proteinkinase assay system.

[0211]FIG. 22 shows the effect of two different staurosporineconcentrations on JNK2α2 activity. The lower concentration had littleeffect, however 5 μM caused approximately 50% inhibition after 30minutes at 30° C.

[0212] The above assay was performed in 200 μl volumes in plastic tubesin a 30° C. waterbath. At each time point 20 μl samples were removedfrom the tubes and added to wells of a 96 well plate, followed by theaddition of 20 μl of ATP detection reagent. The final concentration ofATP used was 12.5 μM, with JNK2α2 at 1.25 μM and ATF-2 at 2.55 μM.

[0213] Genistein

[0214] We also investigated the effect of genistein (Calbiochem) onMAPK-1 activity with MBP as substrate.

[0215] The assay buffer used was the same as for the SAP kinases (seeabove). The assay was performed in 100 μl volumes in a 96 wellmicrotitre plate at 30° C. for 30 minutes. MBP (Calbiochem) was used atthe same concentration as previously described (2.72 μM), with theactivated MAPK1/ERK1 (UBI) used at a final concentration of 2.5 U per100 μl reaction volume. The inhibitor was added at concentrations of 0,0.1, 0.25, 0.5 and 1.0 μM, with 10 μl being added per well in 0.1% (v/v)DMSO.

[0216] For completeness, control samples with and without DMSO (0+DMSOand 0−DMSO, respectively) were also analysed to confirm that there wasno effect of DMSO on the performance of the assay.

[0217]FIG. 23 shows the effect of increasing concentrations of Genistein(in μM) on MAPK-1 activity with MBP as substrate (results are presentedas the means of duplicate wells ±SD). Specifically, the data showed aneffect of genistein at 500 nM, with increased inhibitory activity at 1μM.

[0218] PD098059

[0219] We also investigated the effect of the selective inhibitorPD098059 on the raf-1 activation of inactive MEK-1.

[0220] With PD098059, there was a concentration dependent increase inthe light output in the presence of the inhibitor indicating reducedkinase activity.

[0221]FIG. 24 shows the effect of two different concentrations ofPD098059 on raf-1 activity (results are the means of triplicate wells±SD). These data confirmed the suitability of the assay fordetermination of kinase inhibitory activity.

[0222] This experiment also provides evidence of the versatility ofmethods and assays of the present methods since it was possible todetect activity of another kinase/substrate system, namelyraf-1/inactive MEK-1.

[0223] Appendix 1

[0224] ATP Detection Reagent (ADR) Formulation Reconstituted ADRMagnesium acetate 20 mM Sigma Tetrasodium pyrophosphate 8 μM SigmaBovine Serum Albumin 0.32% w/v Sigma D-Luciferin 712 μM ConCellL-Luciferin 17.8 μM ConCell Luciferase 17 nM Europa Bioproducts Dextran3 mg ml⁻¹ Sigma Tris 40 mM Sigma EDTA 800 μM Sigma

[0225] Final Reaction Concentrations Magnesium acetate 2.36 mMTetrasodium pyrophosphate 236 nM Bovine Serum Albumin 0.009% w/vD-Luciferin 21 μM L-Luciferin 525 nM Luciferase 500 pM Dextran 88.5 gml⁻¹ Tris 1.18 mM EDTA 23.6 μM

[0226] Tris Acetate (TA) buffer (sufficient for 1 liter) Tris  12.1 gSigma EDTA 0.744 g Sigma

[0227] 0.1M Tris, 2 mM EDTA adjust to pH 7.75 with glacial acetic acid.Hepes Buffer 200 mM (sufficient for 1 liter) EDTA 0.744 g Sigma Hepes 47.6 g Sigma

[0228] Adjust to pH 7.75 with glacial acetic acid

[0229] UBI Buffer (as per UBI data sheet)

[0230] 20 mM MOPS pH7.2

[0231] 25 mM β-glycerol phosphate

[0232] 5 mM EGTA

[0233] 1 mM sodium orthovanadate

[0234] 1 mM dithiothreitol JNK Assay Buffer (formulation for × 10concentrate) 250 mM Hepes, pH 7.5 Sigma  1.5M Sodium Chloride Sigma 200mM Magnesium Chloride Sigma 0.01% Tween 20 Sigma

[0235] At time of use there is the addition of 20 mM dithiothreitol(Sigma) and 150 μM ATP (Sigma). ADP Converting Reagent (sufficient for600 mls) Pyruvate kinase (50 000 Units) 20 ml Calbiochem 1M Phosphoenolpyruvate (monosodium salt) 10 ml Sigma 2M Potassium Acetate 100 m SigmaTris acetate buffer pH7.75 470 ml

[0236] Final Concentrations: Reaction Stock Mixture PK 7.6 U/ml 0.8 U/mlPEP 1.67 mM 175 nM Potassium acetate 33 mM 3.5 mM

[0237] Appendix 2: Suppliers

[0238] Berhold Detection systems GmbH

[0239] Bleichstrasse 56-58

[0240] D-75173 Pforzheim

[0241] Germany

[0242] Biltrace Ltd

[0243] The Science Park

[0244] Bridgend

[0245] CF31 3NA

[0246] Calbiochem-Nobabiochem (UK) Ltd

[0247] Boulevard Industrial Park

[0248] Padge Road

[0249] Beeston

[0250] Nottingham NG9 2JR

[0251] ConCell BV

[0252] Wevelinghoven 26

[0253] Nettetal

[0254] D-41334

[0255] Germany

[0256] Europa Bioproducts Ltd

[0257] Europa House

[0258] 15-17 North Street, Wicken

[0259] Ely, Cambridge

[0260] CB7 5XW

[0261] Fahrenheit Lab Supplies

[0262] Northfield Road

[0263] Rotherham

[0264] Dynex Labsystems

[0265] Action Court

[0266] Ashford Road

[0267] Ashford

[0268] Middlesex TW15 1XB

[0269] Labsystems Oy

[0270] Sorvaajankatu 15

[0271] Helsinki

[0272] Finland

[0273] 00810

[0274] Perkin Elmer Life Sciences

[0275] Perkin Elmer House

[0276] 204 Cambridge Science Park

[0277] Cambridge CB4 0GZ

[0278] Sarstedt

[0279] 68 Boston Road

[0280] Beaumont Leys

[0281] Leicester LE4 1AW

[0282] Sigma-Aldrich Co Ltd

[0283] Fancy Road

[0284] Poole

[0285] Dorset BH12 4QH

[0286] Upstate Biotechnology Inc. (UBI)

[0287] 199 Saranac Avenue

[0288] Lake Placid

[0289] NY 12946

1-42. (Canceled)
 43. A method for detecting protein kinase activitycomprising (a) establishing a reaction mixture comprising ATP, a proteinkinase to be tested and a substrate capable of being phosphorylated bythe protein kinase; and (b) using a bioluminescence reaction to detectwhether any change in ATP concentration occurs.
 44. A method fordetermining protein kinase activity comprising (a) establishing areaction mixture comprising ATP, a protein kinase to be tested and asubstrate capable of being phosphorylated by the protein kinase; (b)using a bioluminescence reaction to detect whether any change in ATPconcentration occurs, and (c) using the detection of step (b) to obtaininformation for determining protein kinase activity.
 45. A method foridentifying a compound which modulates the activity of a protein kinase,said method comprising: (a) establishing a reaction mixture comprisingATP, a protein kinase to be tested, a substrate capable of beingphosphorylated by the protein kinase and a compound to be tested for anability to modulate the activity of the protein kinase; (b) using abioluminescence reaction to detect whether any change in ATPconcentration occurs; (c) using the detection of step (b) to obtaininformation for identifying whether the compound modulates the activityof the protein kinase.
 46. The method of claim 43, 44 or 45 wherein thereaction mixture is substantially cell-free.
 47. The method of claim 45wherein the compound to be tested is identified as a protein kinaseinhibitor if the activity of the kinase is lower in the presence of thecompound.
 48. The method of claim 45 wherein the compound to be testedis identified as a protein kinase activator if the activity of thekinase is higher in the presence of the compound.
 49. The method ofclaim 43, 44, or 45 wherein the kinase is activated prior to step (a).50. The method of claim 43, 44 or 45 wherein the reaction mixturecomprises a buffer.
 51. The method of claim 50 wherein the buffer isHepes buffer.
 52. The method of claim 43, 44, or 45 whereinphosphorylation is allowed to proceed at room temperature prior to step(b).
 53. The method of claim 52 comprising a further step (a′), carriedout after step (a) and before step (b), of adding a reagent to thereaction mixture for stopping phosphorylation of the substrate.
 54. Themethod of claim 53 wherein the reagent for stopping phosphorylation isselected from the group consisting of an acid, EGTA and EDTA.
 55. Themethod of claim 54 comprising a further step (a″), carried out afterstep (a′) and before step (b), of adjusting the pH of the reactionmixture formed in step (a∝) to pH 7.0.
 56. The method of claim 55wherein step (a″) comprises adding Hepes buffer.
 57. The method of claim43, 44 or 45 wherein step (b) comprises: (i) adding a bioluminescentreagent comprising luciferin or a derivative thereof and a luciferase tosaid reaction mixture, said luciferin or a derivative thereof emittinglight in a bioluminescent reaction with the luciferase in the presenceof ATP; and (ii) detecting a light intensity, or a change of lightintensity with time, emitted by the bioluminescent reaction.
 58. Themethod of claim 57 wherein step (b) further comprises the followingsteps carried out after the light intensity detected in step (ii) hasreached a substantially constant level: (iii) adding a reagent thatconverts ADP to ATP; (iv) adding a bioluminescent reagent comprisingluciferin or a derivative thereof and luciferase to said reactionmixture of step (iii), said luciferin or a derivative thereof emittinglight in a bioluminescent reaction with the luciferase in the presenceof ATP; and (v) detecting light intensity emitted by the bioluminescentreaction wherein the difference in the intensity of light in step (v)and the steady state intensity of light in step (ii) is a measure of ADPconcentration in the reaction mixture of step (ii).
 59. The method ofclaim 45 wherein the kinase is JNK-1 and the substrate is GST-c-jun. 60.The method of claim 45 wherein the kinase is MAP Kinase-1 (ERK-1) andthe substrate is myelin basic protein.
 61. The method of claim 45wherein the kinase is MAP Kinase-2 (ERK-2) and the substrate is myelinbasic protein.
 62. The method of claim 45 wherein the kinase is PKA andthe substrate is Kemptide.
 63. The method of claim 45 wherein the kinaseis JNK-2 and the substrate is GST-c-jun.
 64. The method of claim 45wherein the kinase is MEK-1 and the substrate is inactive MAP Kinase-2(ERK-2).
 65. The method of claim 45 wherein the kinase is JNKα and thesubstrate is ATF-2.
 66. The method of claim 45 wherein the kinase isJNKα and the substrate is c-jun.
 67. The method of claim 45 wherein thekinase is SAPK-3 and the substrate is myelin.
 68. A kit for detectingprotein kinase activity comprising: (a) a bioluminescent reagentcomprising luciferin or a derivative thereof and a luciferase, saidluciferin or a derivative thereof emitting light in a bioluminescentreaction with the luciferase in the presence of ATP; (b) a kinase; (c) asubstrate capable of being phosphorylated by said kinase; and (d) ATP.69. The kit of claim 68 further comprising one or more buffers forreconstituting, diluting or dissolving the bioluminescent reagent,kinase, substrate and/or ATP.
 70. The kit of claim 68 further comprisinga reagent capable of stopping the reaction of said kinase with saidsubstrate.
 71. The kit of claim 68 further comprising one or morereagent(s) which converts ADP to ATP.
 72. The kit of claim 71, whereinthe reagent which converts ADP to ATP comprises pyruvate kinase andphosphoenol pyruvate.
 73. The kit of claim 68 further comprising two ormore different kinases and corresponding substrates.
 74. The kit ofclaim 68 wherein the reagent or reagents is or are provided inlyophilised form.
 75. The kit of claim 68 further comprising a multiwellmicrotitre plate.
 76. The kit of claim 75 wherein the multiwellmicrotitre plate contains 96 wells or more.
 77. A compound identifiedusing a method according to claim 45.